The identification of body fluids and stains discovered at a crime scene is a major part of forensic investigation today. The three most common fluids found are blood, semen, and saliva, and there are several methods used currently to distinguish one from another. Blood can be presumptively tested for using different color spot tests, but these tests are destructive to the sample and can also have false positives (Siegel, J. A., Ed., “Encyclopedia of Forensic Sciences,” Academic Press, San Diego (2000)). If only a small amount of sample is available, careful decisions must be made as to whether the presumptive test is necessary. There are also confirmatory tests for blood that conclusively prove blood is present, and some of these tests can distinguish between species. Semen is similar in that there are destructive presumptive tests as well as confirmatory tests. Saliva, however, has no confirmatory tests. Therefore, an examiner can never be positive about the presence of saliva (Siegel, J. A., Ed., “Encyclopedia of Forensic Sciences,” Academic Press, San Diego (2000)). Most presumptive tests can be performed in the field, but some sample preparation such as extraction is often necessary. Most confirmatory tests must be done in the laboratory, so forensic experts responding at a crime scene will not know the confirmed identity of fluid traces until much later on. The main problem with these tests is the destruction of the sample. Sometimes a case can be broken with just the smallest amount of biological evidence, so it is crucial that these small quantities are examined as efficiently as possible and nondestructively at the crime scene. Another issue is the ambiguity of the tests. Current simple in-field screening tests do not confirm the presence of a particular fluid, and saliva can never be confirmed. Finally, mixtures of fluids are frequently found, and this can make identification and subsequent DNA analysis even more difficult. The forensic community is in great need of a reliable and ultimately in-field method that can exclusively distinguish between the common and uncommon body fluids, as well as not destroy the sample in the process.
Raman spectroscopy is a technique that is increasing in popularity among the different disciplines of forensic science. Some examples of its use today involve the identification of drugs (Hodges et al., “The Use of Fourier Transform Raman Spectroscopy in the Forensic Identification of Illicit Drugs and Explosives,” Molecular Spectroscopy 46:303-307 (1990)), lipsticks (Rodger et al., “The In-Situ Analysis of Lipsticks by Surface Enhanced Resonance Raman Scattering,” Analyst 1823-1826 (1998)), and fibers (Thomas et al., “Raman Spectroscopy and the Forensic Analysis of Black/Grey and Blue Cotton Fibres Part 1: Investigation of the Effects of Varying Laser Wavelength,” Forensic Sci. Int. 152:189-197 (2005)), as well as paint (Suzuki et al., “In Situ Identification and Analysis of Automotive Paint Pigments Using Line Segment Excitation Raman Spectroscopy: I. Inorganic Topcoat Pigments,” J. Forensic Sci. 46:1053-1069 (2001)) and ink (Mazzella et al., “Raman Spectroscopy of Blue Gel Pen Inks,” Forensic Sci. Int. 152:241-247 (2005)) analysis. The theory behind Raman spectroscopy is based on the inelastic scattering of low-intensity, nondestructive laser light by a solid, liquid or gas sample. Very little or no sample preparation is needed, and the required amount of tested material could be as low as several picograms or femtoliters (10−12 gram or 10−15 liter, respectively). A typical Raman spectrum consists of several narrow bands and provides a unique vibrational signature of the material (Grasselli et al., “Chemical Applications of Raman Spectroscopy,” New York: John Wiley & Sons (1981)). Unlike infrared (IR) absorption spectroscopy, another type of vibrational spectroscopy, Raman spectroscopy shows very little interference from water (Grasselli et al., “Chemical Applications of Raman Spectroscopy,” New York: John Wiley & Sons (1981)), and that makes it a great technique for analyzing body fluids and their traces. Proper Raman spectroscopic measurements do not damage the sample. The stain or swab could be tested on the field and still be available for further use in the lab for DNA analysis, and that is very important to forensic application. The design of a portable Raman spectrometer is a reality now (Yan et al., “Surface-Enhanced Raman Scattering Detection of Chemical and Biological Agents Using a Portable Raman Integrated Tunable Sensor,” Sensors and Actuators B. 6 (2007); Eckenrode et al., “Portable Raman Spectroscopy Systems for Field Analysis,” Forensic Science Communications 3:(2001)) which would lead to the ability to make identifications at the crime scene.
Fluorescence interference is the largest problem with Raman spectroscopy and is perhaps the reason why the latter technique has not been more popular in the past. If a sample contains molecules that fluoresce, the broad and much more intense fluorescence peak will mask the sharp Raman peaks of the sample. There are a few remedies to this problem. One solution is to use deep ultraviolet (DUV) light for exciting Raman scattering (Lednev I. K., “Vibrational Spectroscopy: Biological Applications of Ultraviolet Raman Spectroscopy,” in: V. N. Uversky, and E. A. Permyakov, Protein Structures, Methods in Protein Structures and Stability Analysis (2007)). Practically no condensed face exhibits fluorescence below ˜250 nm. Possible photodegradation of biological samples is an expected disadvantage of DUV Raman spectroscopy. Another option to eliminate fluorescence interference is to use a near-IR (NIR) excitation for Raman spectroscopic measurement. Finally, surface enhanced Raman spectroscopy (SERS) which involves a rough metal surface can also alleviate the problem of fluorescence (Thomas et al., “Raman Spectroscopy and the Forensic Analysis of Black/Grey and Blue Cotton Fibres Part 1: Investigation of the Effects of Varying Laser Wavelength,” Forensic Sci. Int. 152:189-197 (2005)). However, this method requires direct contact with the analyte and cannot be considered to be nondestructive.
There have been other studies performed which involve the analysis of body fluids using Raman spectroscopy. SERS has been used to detect 5-fluorourcil in saliva (Farquharson et al., J. Raman Spectrosc. 36:208-212 (2005)), drugs in blood and urine (Trachta et al., J. Mol. Structure 693:174-185 (2004)), lactic acid in serum (Chiang et al., “Plasmonics: Metallic Nanostructures and Their Optical Properties III,” 5927:1 Z/1-1Z/8 (2005)), and has analyzed body fluids on test strips (U.S. Patent Publication No. 2007/0224683 A1 to Clarke et al.). It has also been applied to the in vitro detection of analytes in body fluids (U.S. Patent Publication No. 2006/0240401 A1 to Clarke et al.), the detection of low levels of body fluids (Kindcade, K., Laser Focus World 42:109-111 (2006)), and has been used as part of a probe system to detect protein analytes (U.S. Patent Publication No. 2005/0148100 A1 to Su et al.). Many other Raman experiments not involving SERS have been conducted for detecting cancer (U.S. Patent Publication No. 2006/0170928 to Masilamani et al.), characterizing whole blood (Sato et al., J. Biomed. Opt. 6:366-370 (2001)), urine (Premasiri et al., Lasers Surg. Med. 28:330-334 (2001)), in vivo fluids (WO2006061565 to Matousek et al.), breath samples (WO2006136281 to Wolfgang), and amniotic fluid (U.S. Patent Publication No. 2006/0247536 A1 to Koski et al.). Body fluids such as urine, blood, blood plasma, blood serum, saliva, and sweat have been spiked with various components, and these components have been analyzed simultaneously by multivariate regression analysis (U.S. Pat. No. 5,796,476 to Wang et al.). One goal of the invention is to evaluate the potential use of NIR Raman spectroscopy for confirmatory analysis of body fluids for forensic purposes.
Other spectroscopic techniques have been also tested for the non-destructive analysis of body fluids. Fluorescence has been used to detect biological materials (U.S. Pat. No. 6,750,006 to Powers et al.) and microorganisms (Estes et al, “Reagentless Detection of Microorganisms by Intrinsic Fluorescence,” Biosens. Bioelectron. 18:511-519 (2003)). Photoluminescence has also been used in the form of a light source known as Polilight to detect stains (Jackson et al., “The Use of Polilight in the Detection of Seminal Fluid, Saliva, and Bloodstains and Comparison with Conventional Chemical-Based Screening Tests,” J. Forensic Sci. 51(2):361-70 (2006) and J. Forensic Sci. 52:740-41 (2007); Stoilovic, M., “Detection of Semen and Blood Stains Using Polilight as a Light Source,” Forensic Sci. Int. 51:289-296 (1991)), and this source has been compared to Luma-Light and Spectrum 9000 (Watkin et al., “A Comparison of the Forensic Light Sources, Polilight, Luma-Light, and Spectrum 9000,” J. Forensic Identification 44:632 (1994)). Ultra-violet light has been used to enhance blood (Klasey et al., “Using Ultra-Violet Light to Enhance Blood,” J. Forensic Identification 42:404 (1992)), and body secretions have been analyzed with high intensity quartz arc tubes (Auvdel M. J., “Comparison of Laser and High-Intensity Quartz Arc Tubes in the Detection of Body Secretions,” J. Forensic Sci. 33:929-945 (1988)). It is a general advantage of Raman spectroscopy over photoluminescence in providing more specific information about the analyte. As a result, one could expect that Raman spectroscopy should offer a higher identification power when applied for body fluid identification especially in the case of mixed and contaminated samples.
Accordingly, the present invention is directed to overcoming these deficiencies in the art.